This specification claims the benefit of PCT/GB/01206, filed Apr. 24, 1998, and GB Application 9708479.2, filed Apr. 25, 1997.
This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABAA receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.
Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABAA receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and xcex2-carbolines, in addition to sites for the agonist ligand GABA (for reviews see MacDonald and Olsen, Ann. Rev. Neurosci., 1994, 17, 569; and Whiting et al, Int. Rev. Neurobiol., 1995, 38, 95).
Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (xcex1, xcex2, xcex3 and xcex4) based on their sequence similarities. To date, in mammals, six types of xcex1 (Schofield et al., Nature (London), 1987, 328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al., EMBO J., 1989, 8, 1665; Pritchett and Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatisky et al., Neuron, 1989, 3, 745), three types of xcex2 (Ymer et al., EMBO J., 1989, 8, 1665), three types of xcex3 (Ymer et al., EMBO J., 1990, 9, 3261; Shivers et al., Neuron, 1989, 3, 327: and Knoflach et al, FEBS Lett., 1991, 293, 191) and one xcex4 subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified. More recently, a further member of the GABA receptor gene family, xcex5, has been identified (Davies et al, Nature, 1997, 385, 820). The polypeptide is 506 amino acids in length and exhibits greatest amino acid sequence identity with the GABAA receptor xcex33 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth xcex3 subunit.
The differential distribution of many of the subunits has been characterised by in situ hybridisation (Shivers et al., Neuron, 1989, 3, 327; Wisden et al, J. Neurosci., 1992, 12, 1040; and Laurie et al, J. Neurosci, 1992, 12, 1063) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex.
Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABAA receptors in vivo (Pritchett et al., Science, 1989, 245, 1389; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; Luddens et al., Nature (London), 1990, 346, 648; Hadingham et al, Mol. Pharmacol., 1993, 43, 970; and Hadingham et al., Mol. Pharmacol., 1993, 44, 1211). This approach has revealed that, in addition to an xcex1 and xcex2 subunit, either xcex31 or xcex32 (Pritchett et al., Nature (London), 1989, 338, 582; Ymer et al., EMBO J., 1990, 9, 3261; and Wafford et al., Mol. Pharmacol., 1993, 44, 437) or xcex33 (Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; and Wilson-Shaw et al., FEBS Lett., 1991, 284, 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the xcex1 and xcex3 subunits present. Receptors containing a xcex4 subunit (i.e. xcex1xcex3xcex4) do not appear to bind benzodiazepines (Shivers et al., Neuron, 1989, 3, 327; and Quirk et al., J. Biol. Chem., 1994, 269, 16020). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ1 type receptor (xcex11xcex21xcex32) and a BZ2 type receptor (xcex12xcex21xcex32 or xcex13xcex21xcex32, Pritchett et al., Nature (London), 1989, 338, 582), as well as GABAA receptors with a novel pharmacology, xcex15xcex22xcex32 (Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802), xcex14xcex22xcex32 (Wisden et al, FEBS Lett., 1991, 289, 227) and xcex16xcex22xcex32 (Luddens et al., Nature (London), 1990, 346, 648). The pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, and biochemical expperiments have begun to determine the subunit composition of native GABA receptors (McKernan and Whiting, Tr. Neurosci., 1996, 19, 139). The exact structure of receptors in vivo has yet to be definitively elucidated.
The present invention relates to a new class of GABA receptor subunit, hereinafter referred to as the theta subunit (xcex8 subunit).
The nucleotide sequence for the theta subunit, together with its deduced amino acid sequence corresponding thereto, is depicted in FIG. 1 of the accompanying drawings.
The present invention accordingly provides, in a first aspect, a DNA molecule encoding the theta subunit of the human GABA receptor comprising all or a portion of the sequence depicted in FIG. 1, or a modified human sequence.
In an alternative aspect, the present invention provides a DNA molecule encoding the theta subunit of the human GABA receptor comprising all or a portion of the sequence depicted in FIG. 2, or a modified human sequence.
The term xe2x80x9cmodified human sequencexe2x80x9d as used herein referes to a variant of the DNA sequences depicted in FIG. 1 and FIG. 2. Such variants may be naturally occuring allelic variants or non-naturally occuring or xe2x80x9cengineeredxe2x80x9d variants. Allelic variation is well known in the art in which the nucleotide sequence may have a substitution, deletion or addition of one or more nucleotides without substantial alteration of the function of the encoded polypeptide. Particularly preferred allelic variants arise from nucleotide substitution based on the degeneracy of the genetic code.
The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 1; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989.
In a further aspect, the present invention also relates to polynucleotides (for example, cDNA, genomic DNA or synthetic DNA) which hybridize under stringent conditions to the DNA molecules depicted in FIG. 1 and FIG. 2. As used herein, the term xe2x80x9cstringent conditionsxe2x80x9d will be understood to require at least 95% and preferably at least 97% identity between the hybridized sequences. Polynucleotides which hybridize under stringent conditions to the DNA molecules depicted in FIG. 1 and FIG. 2 preferably encode polypeptides which exhibit substantially the same biological activity or function as the polypeptides depicted in FIG. 1 and FIG. 2, respectively.
The present invention further relates to a GABA theta subunit polypeptide which has the deduced amino acid sequence of FIG. 1 or FIG. 2, as well as fragments, analogs and derivatives thereof.
The terms xe2x80x9cfragmentxe2x80x9d, xe2x80x9cderivativexe2x80x9d and xe2x80x9canalogxe2x80x9d when referring to the polypeptide of FIG. 1 or FIG. 2, means a polypeptide which retains essentially the same biological activity or function as the polypeptide depicted in FIG. 1 or FIG. 2. Thus, an analog may be, for example, a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of FIG. 1 or FIG. 2 may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residues may or may not be one encoded by the genetic code; or one in which one or more of the amino acid residues includes a substituent group; or one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the technical capabilities of those skilled in the art.
The polypeptides and DNA molecules of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term xe2x80x9cisolatedxe2x80x9d means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring DNA molecule or polypeptide present in a living animal is not isolated, but the same DNA molecule or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such DNA molecules could be part of a vector and/or such DNA molecules or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
In another aspect, the invention provides a recombinant expression vector comprising the nucleotide sequence of the human GABA receptor theta subunit together with additional sequences capable of directing the synthesis of the said human GABA receptor theta subunit in cultures of stably co-transfected eukaryotic cells.
The term xe2x80x9cexpression vectorsxe2x80x9d as used herein refers to DNA sequences that are required for the transcription of cloned copies of recombinant DNA sequences or genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, yeast cells, insect cells, plant cells and animal cells. Specifically designed vectors allow the shuttling of DNA between bacteria-yeast, bacteria-plant or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
The term xe2x80x9ccloning vectorxe2x80x9d as used herein refers to a DNA molecule, usually a small plasmid or bacteriophage DNA capable of self-replication in a host organism, and used to introduce a fragment of foreign DNA into a host cell. The foreign DNA combined with the vector DNA constitutes a recombinant DNA molecule which is derived from recombinant technology. Cloning vectors may include plasmids, bacteriophages, viruses and cosmids.
The recombinant expression vector in accordance with the invention may be prepared by inserting the nucleotide sequence of the GABA theta subunit into a suitable precursor expression vector (hereinafter referred to as the xe2x80x9cprecursor vectorxe2x80x9d) using conventional recombinant DNA methodology known from the art. The precursor vector may be obtained commercially, or constructed by standard techniques from known expression vectors. The precursor vector suitably contains a selection marker, typically an antibiotic resistance gene, such as the neomycin or ampicillin resistance gene. The precursor vector preferably contains a neomycin resistance gene, adjacent the SV40 early splicing and polyadenylation region; an ampicillin resistance gene; and an origin of replication, e.g. pBR322 ori. The vector also preferably contains an inducible promoter, such as MMTV-LTR (inducible with dexamethasone) or metallothionin (inducible with zinc), so that transcription can be controlled in the cell line of this invention. This reduces or avoids any problem of toxicity in the cells because of the chloride channel intrinsic to the GABAA receptor.
One suitable precursor vector is pMAMneo, available from Clontech Laboratories Inc. (Lee et al., Nature, 1981, 294, 228; and Sardet et al., Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneo can be constructed from the vectors pMSG and pSV2neo.
The recombinant expression vector of the present invention is then produced by cloning the GABA receptor theta subunit cDNA into the above precursor vector. The receptor subunit cDNA is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the Hind III site, in the polylinker of the precursor vector, for example pMAMneo or pMSGneo, by standard cloning methodology known from the art, and in particular by techniques analogous to those described herein. Before this subdoning, it is often advantageous, in order to improve expression, to modify the end of the theta subunit cDNA with additional 5xe2x80x2 untranslated sequences, for example by modifying the 5xe2x80x2 end of the theta subunit DNA by addition of 5xe2x80x2 untranslated region sequences from the xcex11 subunit DNA. Alternatively, expression of the theta subunit cDNA may be modified by the insertion of an epitope tag sequence such as c-myc.
According to a further aspect of the present invention, there is provided a stably co-transfected eukaryotic cell line capable of expressing a GABA receptor, which receptor comprises the theta receptor subunit, at least one alpha receptor subunit and optionally one or more beta, gamma, delta, or epsilon receptor subunit.
This is achieved by co-transfecting cells with multiple expression vectors, each harbouring cDNAs encoding for an xcex1, xcex8, and optionally one or more xcex2, xcex3, xcex4 , or GABA receptor subunits. In a further aspect, therefore, the present invention provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA receptor, which comprises stably co-transfecting a eukaryotic host cell with at least two expression vectors, one such vector harbouring the cDNA sequence encoding the theta GABA receptor subunit, and another such vector harbouring the cDNA sequence encoding an alpha GABA receptor subunit. The stable cell-line which is established expresses an xcex1xcex8 GABA receptor.
Each receptor thereby expressed, comprising a unique combination of xcex1, xcex8 and optionally one or more subunits selected from xcex2, xcex3, xcex4 or xcex4 subunits, will be referred to hereinafter as a GABA receptor xe2x80x9csubunit combinationxe2x80x9d.
Expression of the GABA receptor may be accomplished by a variety of different promoter-expression systems in a variety of different host cells. The eukaryotic host cells suitably include yeast, insect and mammalian cells. Preferably the eukaryotic cells which can provide the host for the expression of the receptor are mammalian cells. Suitable host cells include rodent fibroblast lines, for example mouse Ltkxe2x88x92, Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; and HEK293 cells. It is necessary to incorporate at least one a subunit, the xcex8 subunit, and optionally one or more subunits selected from xcex2, xcex3xcex4 or xcex4 into the cell line in order to produce the required receptor. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for.
In order to employ this invention most effectively for screening purposes, it is preferable to build up a library of cell lines, each with a different combination of subunits. Typically a library of 5 or 6 cell line types is convenient for this purpose. Preferred subunit combinations include: xcex1xcex8xcex2, xcex1xcex8xcex3, xcex1xcex8xcex4, and xcex1xcex8xcex5, and most especially xcex11xcex8xcex32. Further preferred subunit combinations include xcex1xcex2xcex8xcex3 and xcex1xcex2xcex8xcex5, and most especially xcex12xcex21xcex8xcex31 and xcex12xcex23xcex8xcex32.
Cells are then co-transfected with the desired combination of the expression vectors. There are several commonly used techniques for transfection of eukaryotic cells in vitro. Calcium phosphate precipitation of DNA is most commonly used (Bachetti et al., Proc. Natl. Acad. Sci. USA, 1977, 74, 1590-1594; Maitland et al., Cell, 1977, 14, 133-141), and represents a favoured technique in the context of the present invention.
A small percentage of the host cells takes up the recombinant DNA. In a small percentage of those, the DNA will integrate into the host cell chromosome. Because an antibitotic resistance marker gene, such as the neomycin or zeocin resistance gene, will have been incorporated into these host cells, they can be selected by isolating the individual clones which will grow in the presence of the chosen antibiotic, e.g. neomycin or zeocin. Each such clone may then tested to identify those which will produce the receptor. This may be achieved by inducing the production, for example with dexamethasone, and then detecting the presence of receptor by means of radioligand binding.
Alternatively, expression of the GABA receptor may be effected in Xenopus oocytes (see, for instance, Hadingham et al. Mol. Pharmacol., 1993, 44, 1211-1218). Briefly, isolated oocyte nuclei are injected directly with injection buffer or sterile water containing at least one alpha subunit, the theta subunit, and optionally one or more beta, gamma, delta or epsilon receptor subunits, engineered into a suitable expression vector. The oocytes are then incubated.
The expression of subunit combinations in the transfected oocytes may be demonstrated using conventional patch clamp assay. This assay measures the charge flow into and out of an electrode sealed on the surface of the cell. The flow of chloride ions entering the cell via the GABA gated ion channel is measured as a function of the current that leaves the cell to maintain electrical equilibrium within the cell as the gate opens.
In a further aspect, the present invention provides protein preparations of GABA receptor subunit combinations, especially human GABA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells.
The protein preparations of GABA receptor subunit combinations can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The GABA theta subunit polypeptide of the present invention is also useful for identifying other subunits of the GABA receptor. An example of a procedure for identifying these subunits comprises raising high titre polyclonal antisera against unique, bacterially expressed GABA theta polypeptides. These polyclonal antisera are then used to immunoprecipitate detergent-solubilized GABA receptors from a mammalian brain, for example, a rat brain.
The invention also provides preparations of membranes containing subunit combinations of the GABA receptor, especially human GABA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells.
The cell line, and the membrane preparations therefrom, according to the present invention have utility in screening and design of drugs which act upon the GABA receptor, for example benzodiazepines, barbiturates, xcex2-carbolines and neurosteroids.
Receptor localisation studies using in situ hybridization in monkey brains shows that the xcex8 subunit has a restricted localisation; residing mainly in components of the limbic system (involved in emotions such as rage, fear, motivation sexual behaviours and feeding); medial septum, cingulate cortex, the amygdala and hippocampal fields, in various hypothalamic nuclei, and in regions that have been associated with pain perception; the cingulate cortex, insular cortex, and in mid brain and pons structures.
The present invention accordingly provides the use of stably cotransfected cell lines described above, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon GABA receptors comprising the xcex8 subunit. Of particular interest in this context are molecules capable of interacting selectively with GABA receptors made up of varying subunit combinations. As will be readily apparent, the cell line in accordance with the present invention, and the membrane preparations derived therefrom, provide ideal systems for the study of structure, pharmacology and function of the various GABA receptor subtypes. In particular, preferred screens are functional assays utilizing the pharmacological properties of the GABA receptor subunit combinations of the present invention.
Thus, according to a further aspect of the present invention, there is provided a method for determining whether a ligand, not known to be capable of binding to a human GABAA receptor comprising the theta subunit, can bind to a human GABAA receptor comprising the theta subunit, which comprises contacting a mammalian cell comprising DNA molecules encoding at least one alpha receptor subunit, the theta receptor subunit, and optionally one or more beta, gamma, delta or epsilon receptor subunits with the ligand under conditions permitting binding of ligands known to bind to the GABAA receptor, detecting the presence of any of the ligand bound to the GABAA receptor comprising the theta subunit, and thereby determining whether the ligand binds to the GABAA receptor comprising the theta subunit. The theta subunit-encoding DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1 or FIG. 2. Preferably, the mammalian cell is non-neuronal in origin. An example of a non-neuronal mammalian cell is a fibroblast cell such as an Ltkxe2x88x92cell. The preferred method for determining whether a ligand is capable of binding to a human GABAA receptor comprising the theta subunit comprises contacting a transfected non-neuronal mammalian cell (i.e. a cell that does not naturally express any type of GABAA receptor, and thus will only express such a receptor if it is transfected into the cell) expressing a GABAA receptor comprising the theta subunit on its surface, or contacting a membrane preparation from such a transfected cell, with the ligand under conditions which are known to prevail, and thus to be associated with, in vivo binding of the ligands to a GABAA receptor comprising the theta subunit, detecting the presence of any of the ligand being tested bound to the GABAA receptor comprising the theta subunit on the surface of the cell, and thereby determining whether the ligand binds to a human GABAA receptor comprising the theta subunit. This response system may be based on ion flux changes measured, for example, by scintillation counting (where the ion is radiolabelled) or by interaction of the ion with a fluorescent marker. Particularly suitable ions are chloride ions. Such a host system is conveniently isolated from pre-existing cell lines. Such a transfection system provides a complete response system for investigation or assay of the activity of human GABAA receptors comprising the theta subunit with ligands as described above. Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for these competitive binding assays. A transfection system constitutes a xe2x80x9cdrug discovery systemxe2x80x9d useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate, inhibit or modulate the natural functions of human GABAA receptors comprising the theta subunit. The transfection system is also useful for determining the affinity and efficacy of known drugs at human GABAA receptor sites comprising the theta subunit.
This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a human GABAA receptor comprising the theta subunit on the surface of a cell which comprises contacting a mammalian cell comprising DNA molecules encoding at least one alpha receptor subunit, the theta receptor subunit and optionally one or more beta, gamma, delta or epsilon receptor subunits on the surface of a cell with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, human GABAA receptors comprising the theta subunit. The theta subunit-encoding DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1 or FIG. 2. Preferably, the mammalian cell is non-neuronal in origin. An example of a non-neuronal mammalian cell is a fibroblast cell such as an Ltkxe2x88x92cell. Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed GABAA receptor protein in transfected cells, using radioligand binding methods well known in the art. Drug candidates are also screened for selectivity by identifying compounds which bind with high affinity to one particular GABAA receptor combination but do not bind with high affinity to any other GABAA receptor combination or to any other known receptor site. Because selective, high affinity compounds interact primarily with the target GABAA receptor site after administration to the patient, the chances of producing a drug with unwanted side effects are minimized by this approach.
In the above screens, the mammalian cell may, for example, comprise DNA molecules encoding at least one alpha receptor subunit, the theta subunit, and optionally one or more gamma receptor subunits and optionally one or more beta receptor subunits.
More preferably, in the above screens, the mammalian cell comprises DNA molecules encoding at least one alpha receptor subunit, at least one gamma receptor subunit and the theta receptor subunit.
Ligands or drug candidates identified above may be agonists or antagonists at human GABAA receptors comprising the theta subunit, or may be agents which allosterically modulate a human GABAA receptor comprising the theta subunit. These ligands or drug candidates identified above may be employed as therapeutic agents, for example, for the modulation of emotions such as rage and fear, of sexual and appetite behaviours and of pain perception.
The ligands or drug candidates of the present invention thus identified as therapeutic agents may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the agonist or antagonist, and a pharmaceutically acceptable carrier or excipient.
Preferably the compositions containing the ligand or drug candidate identified according to the methods of the present invention are in unit dosage forms such as tablets, pills, capsules, wafers and the like. Additionally, the therapeutic agent may be presented as granules or powders for extemporaneous formulation as volume defined solutions or suspensions. Alternatively, the therapeutic agent may be presented in ready-prepared volume defined solutions or suspensions. Preferred forms are tablets and capsules.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil or soybean oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
Compositions of the present invention may also be administered via the buccal cavity using conventional technology, for example, absorption wafers.
Compositions in the form of tablets, pills, capsules or wafers for oral administration are particularly preferred.
A minimum dosage level for the ligand or drug candidate identified according to the methods of the present invention is about 0.05 mg per day, preferably about 0.5 mg per day and especially about 2.5 mg per day. A maximum dosage level for the ligand or drug candidate is about 3000 mg per day, preferably about 1500 mg per day and especially about 500 mg per day. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily, and especially once a day.
It will be appreciated that the amount of the therapeutic agent required for use therapy will vary not only with the particular compounds or compositions selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient""s physician or pharmacist.